Experiencing the micro-world - a cell's perspective

Lead Research Organisation: Heriot-Watt University
Department Name: Sch of Engineering and Physical Science

Abstract

In the body, most cells grow in close contact with other neighbouring cells and with a local matrix of proteins and sugars that combine to provide an instructive microenvironment. Until recently, most research labs (in both academic and industrial settings) have used 2D cultures of cells on plastic to study cell behaviour, a significant departure from what is actually happening in vivo that can limit the applicability of their research. However, there has been a recent and dramatic shift away from traditional 2D culture to the use of complex, 3D cultures, that more effectively mimic the micro-environment experienced by cells in vivo. This development impacts directly on fields such as regenerative medicine, drug discovery and cancer research, with significant opportunities for improved in vitro modelling of cell behaviour. Despite these improvements in culture techniques, the interaction of the cells with their local microenvironment - a key target in therapies for cancer, wound healing, and fibrosis etc. - remains a 'black box' with technologies unable investigate these environments at the cell level. This proposal will 'open that box', developing the technology and methodology urgently required to fully explore 3D cell cultures on length scales comparable, or smaller than, single cells.

The currently accepted protocol to characterise natural and synthetic matrices, uses a bulk rheometer to produce a single, averaged value of the viscosity and elasticity of the material, destroying the sample in the process. Information about the matrix local to the cells growing inside the samples is lost. Our vision is to image and characterise 3D cell culture environments in all three spatial dimensions, over an extended time course, and on a single multifunctional instrument so that the information can be integrated and mapped. To achieve this we will develop a minimally-invasive technique to measure the 3D micro-rheology of the extracellular matrix using nano- (smaller than the cells) and micro-sized (can be the same size at the cells) beads as local probes. These probes will be held at a fixed position within the matrix using an optical trap and their Brownian motion in all three spatial dimensions tracked using multiplane imaging. The micro-rheology (viscosity and elasticity) of the extracellular matrix local to the probe is extracted from temporal analysis of the Brownian motion. To achieve deep 4D (x,y,z, time) images of live 3D cell cultures, we will combine light sheet microscopy with adaptive optics (a technique for correcting for sample aberrations that reduce image quality deep into complex samples). The final multifunctional platform will be the exciting culmination of these 4 microscopy techniques - optical trapping, multiplane imaging, light sheet microscopy and adaptive optics - capable of imaging and micro-mechanically sensing the 3D environment close to cells.

The output from this work will be the innovation required to allow scientists to study how cells interact with their local microenvironment, combining technologies in a way that's not been possible previously, to observe both the cells, and the forces they exert and are responding to, as they grow and move in 3D space over time. The ability to study cell behaviour in this way is of importance for developing therapies for diseases where cells respond abnormally to signals from their local matrix, such as cancer, providing targets for new drug design. We will include a demonstration of how this can work in our study using both traditional anti-cancer drugs and more innovative therapies such as functionalised nanoparticles. We anticipate that the technology will be useful to both academics and industry (particularly drug discovery in the pharmaceutical industry) and we will work closely with these groups throughout the course of this project to ensure that, once proven, this technology can work for them.

Publications

10 25 50
 
Description This work aims to develop a new instrument capable of imaging cells live in 3D culture over a long time course whilst mapping to these images local readings of the viscosity and elasticity of the matrix next to the cells. Our progress so far has been to build a novel light sheet microscope that is fully beam scanning and the sample remains stationary throughout imaging. This has allowed us to image the same 3D cluster of live cells over a 5 day period. In parallel we have developed a new approach to optical trapping micro-rheology that allows the viscosity and elasticity to be characterised in 3D as opposed to the 2D that was previously possible. We have published data that demonstrates our ability to use micro-rheology methodologies to characterise the material properties of peptide hydrogel comparing our readings to those taken with a more traditional oscillatory rheometer. Working on this project are 4 researchers (3 PDRAs and 1 technician) who are developing important multi-disciplinary skills working with a project team that spans Medicine, Engineering, Physics and Pharmacy. New research questions have opened up in collaboration with the University of Lincoln in Marine Biology studying the local viscosity gradients surrounding plankton.
Exploitation Route Not yet applicable.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Fully humanised 3D vascular perfused model for breast cancer modelling and therapeutic screening
Amount £75,911 (GBP)
Funding ID NC/T001259/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2019 
End 09/2021
 
Description Lighting the Way to a Healthy Nation - Optical 'X-rays' for Walk Through Diagnosis & Therapy
Amount £5,577,754 (GBP)
Funding ID EP/T020997/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 05/2020 
End 05/2025
 
Description Replacing the need for patient-derived xenografts and matrigel organoid culture as preclinical models for breast cancer
Amount £75,591 (GBP)
Funding ID NC/T001267/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2019 
End 06/2020
 
Description U-care: Deep ultraviolet light therapies
Amount £6,132,366 (GBP)
Funding ID EP/T020903/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 05/2020 
End 06/2025
 
Description Understanding How Matrix Remodelling & Respiratory Infection Impact Progression Of Idiopathic Pulmonary Fibrosis Using Stem Cell Derived Alveolar Cells
Amount £93,000 (GBP)
Organisation British Lung Foundation (BLF) 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2019 
End 08/2022
 
Title Peptide gels for 3D breast cancer models 
Description Current materials used for in vitro disease models are often limited by their poor similarity to human tissue, batch-to-batch variability and high complexity in composition and manufacture. We have developed a "blank slate" culture environment that can be customized by incorporating matrix components specifically selected to match the target tissue, with mechanical properties controlled independently and simultaneously. Based on a self-assembling peptide hydrogel, this system contains no exogenous proteins or glycosaminoglycans: only those specifically added, or those synthesized by the cells in culture. This 3D culture platform therefore provides full control over biochemical and physical properties, allowing the composition and mechanics of the tissue of interest to be recapitulated in vitro. As proof-of-concept, we designed a panel of hydrogels designed to mimic the stages of breast cancer progression. Controlling the peptide gelator concentration allows hydrogel stiffness to be matched to normal breast (<1 kPa) or breast tumour (>1 kPa), with higher stiffness favouring the viability of breast cancer cells over normal breast cells. In parallel, these hydrogels may be modified with matrix components relevant to human breast, such as collagen I and hyaluronan. The choice and concentration of these additions control the size, shape and organisation of the breast epithelial cell structures formed in co-culture with fibroblasts. This system therefore provides a means of unravelling the individual influences of matrix, mechanical properties and cell-cell interactions in cancer and disease. 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2018 
Provided To Others? Yes  
Impact We have presented the gel technology at events specifically designed to encourage uptake by other researchers working in this area (e.g. BACR meeting in Leeds, May 2018) and have trained researchers from other groups to encourage uptake of the technology. These training visits were supported financially by NC3Rs. We have also developed additional collaborations related to this technology including with a commercial partner able to provide the raw materials required for the gels. By demonstrating the applicability of the peptide gels to breast cancer modelling we have also attracted additional collaborations with groups working on fibrosis and the support of stem cell differentiation. 
 
Description Adelaide Nottingham Joint PhD Studentship 
Organisation University of Adelaide
Country Australia 
Sector Academic/University 
PI Contribution A new joint PhD student started in January 2021. The student will be supervised by Kylie Dunning and Amanda Wright and will spend years 1 and 3 in Adelaide and year 2 in Nottingham
Collaborator Contribution Initial contact was made via Kylie Dunning and we then applied jointly for this studentship.
Impact PhD studentship
Start Year 2020
 
Description Joint PhD student with Kishan Dholakia 
Organisation University of St Andrews
Country United Kingdom 
Sector Academic/University 
PI Contribution We have a joint student under the Nottingham and Adelaide Studentship scheme
Collaborator Contribution With Kylie Dunning at the University of Adelaide we were awarded a joint PhD studentship via the Nottingham and Adelaide partnership. Kishan Dholakia is on secondment at the University of Adelaide and is part of the supervisory team. The student started in January 2021.
Impact PhD student started in January 2021
Start Year 2020
 
Description School talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 4th December 2019: Lynn Paterson gave a talk to around 30 students from Kinross High School, about her research 'Shedding new light on biology' and spent 5 minutes of the 1 hour talk discussing the ideas around our nusense project.
Year(s) Of Engagement Activity 2019
 
Description School talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 13th November 2019: Lynn Paterson gave a talk to around 70 students from Grantown Grammar School, Grantwon on Spey, around her research area 'Shedding new light on biology' and spent 5 minutes of the 1 hour talk discussing the ideas around our nusense project.
Year(s) Of Engagement Activity 2019